Age-Related Inflammation Triggers Skeletal Stem/Progenitor Cell

Age-related inflammation triggers skeletal stem/ progenitor cell dysfunction

Anne Marie Josephsona,b, Vivian Bradaschia-Correaa, Sooyeon Leea, Kevin Leclerca, Karan S. Patela, Emma Muinos Lopeza, Hannah P. Litwaa, Shane S. Neibarta, Manasa Kadiyalaa, Madeleine Z. Wonga, Matthew M. Mizrahia, Nury L. Yima, Austin J. Rammea, Kenneth A. Egola, and Philipp Leuchta,b,1

aDepartment of Orthopedic Surgery, New York University School of Medicine, New York, NY 10003; and bDepartment of Cell Biology, New York University School of Medicine, New York, NY 10016

Edited by Helen M. Blau, Stanford University, Stanford, CA, and approved February 25, 2019 (received for review June 21, 2018) Aging is associated with impaired tissue regeneration. Stem cell Results number and function have been identified as potential culprits. We Skeletal Stem Cell Frequency Decreases with Aging. To investigate a first demonstrate a direct correlation between stem cell number and clinically relevant age-associated effect on skeletal stem cell fre- time to bone fracture union in a human patient cohort. We then quency and function, we first examined iliac crest bone graft devised an animal model recapitulating this age-associated decline in (ICBG) samples from 36 patients (20 male, 16 female) with ages bone healing and identified increased cellular senescence caused by a ranging from 24 to 89 y who underwent operative fixation of an systemic and local proinflammatory environment as the major contrib- upper- or lower-extremity fracture. FACS with CD271 as a human utor to the decline in skeletal stem/progenitor cell (SSPC) number and skeletal stem cell marker (8–11) revealed that SSPC frequency function. Decoupling age-associated systemic inflammation from chro- significantly declined with increasing age (Fig. 1 A and B). It is Nfkb1 nological aging by using transgenic KO mice, we determined generally well accepted among orthopedic surgeons that fractures in that the elevated inflammatory environment, and not chronological elderly subjects heal more slowly and less reliably, and therefore we age, was responsible for the decrease in SSPC number and function. asked whether SSPC frequency correlates with time to bony union. By using a pharmacological approach inhibiting NF-κB activation, we We prospectively evaluated clinical and radiographic fracture union demonstrate a functional rejuvenation of aged SSPCs with decreased in this cohort and discovered that a lower SSPC number was as- senescence, increased SSPC number, and increased osteogenic function. sociated with longer time to fracture union (Fig. 1C). To identify the MEDICAL SCIENCES Unbiased, whole-genome RNA sequencing confirmed the reversal of mechanism involved in this decline in SSPC number and function, the aging phenotype. Finally, in an ectopic model of bone healing, we we chose a mouse model to further investigate the process of demonstrate a functional restoration of regenerative potential in aged SSPCs. These data identify aging-associated inflammation as the cause skeletal stem cell aging. of SSPC dysfunction and provide mechanistic insights into its reversal. Aging Impairs Bone Regeneration. To evaluate the extent to which the process of aging affects bone healing, we first employed a regeneration | skeletal stem cell | senescence | inflammation | standardized tibial monocortical defect model in young (12-wk- bone healing old) and middle-aged (52-wk-old) male C57BL/6 mice. We analyzed bone healing by using histology, histomorphometry, and ll tissues are affected by aging, but diseases that weaken the micro-CT (μCT). Two weeks after surgery, the injury sites were Askeleton constitute the most prevalent chronic impairment analyzed by histology. Whereas injuries in the young animals in the United States (1). Although skeletal diseases and conditions showed abundant woven bone within the defect site (Fig. 2 A and are seldom fatal, they can significantly compromise function and diminish quality of life. Perhaps most importantly, age-related Significance changes in skeletal health may be traced back to the skeletal stem cell. Like other stem-cell pools, skeletal stem/progenitors are As we age, our capacity for tissue repair and regeneration in impacted by aging. For example, skeletal stem cells from people response to injury declines. Accordingly, bone repair is delayed older than age 65 y, even if they are healthy, make less bone than and impaired in older patients. At the cornerstone of bone stem cells from younger individuals, irrespective of sex (2). Instead healing is the skeletal stem/progenitor cell (SSPC), whose func- of becoming bone-producing osteoblasts, skeletal stem cells from tion and number diminishes with age. However, the mechanisms older people differentiate into fat-producing adipocytes (3), and driving this decline remain unclear. Here, we identify age- this may partly explain why bone-forming ability declines with associated inflammation (“inflamm-aging”)asthemainculpritof increasing age (3, 4). SSPC dysfunction and provide support for a central role of NF-κB Chronic inflammation in the elderly (“inflamm-aging”) is thought as a mediator of inflamm-aging. Our results show that modifica- to be a major contributor to the decline in the regenerative capacity tion of the inflammatory environment may be a translational ap- of the skeleton (5). In contrast to a well-balanced inflammatory re- proach to functionally rejuvenate the aged SSPC, thereby sponse after trauma, which is crucial for successful bone repair (6), improving the regenerative capacity of the aged skeleton. chronic unbalanced elevation of proinflammatory cytokines inhibits regeneration in a variety of other tissues (7). Its effect on the skeletal Author contributions: A.M.J., V.B.-C., S.L., K.L., and P.L. designed research; A.M.J., V.B.-C., S.L., K.L., K.S.P., E.M.L., H.P.L., S.S.N., M.K., M.Z.W., M.M.M., N.L.Y., A.J.R., K.A.E., and P.L. stem/progenitor cell (SSPC) is yet unknown. To address this performed research; A.M.J., V.B.-C., S.L., K.L., K.S.P., E.M.L., H.P.L., and P.L. analyzed data; knowledge gap, we hypothesized that chronic inflammation me- and A.M.J. and P.L. wrote the paper. diated by NF-κB activation—irrespective of age—contributes to a The authors declare no conflict of interest. deterioration of the regenerative function of the stem-cell pool by This article is a PNAS Direct Submission. inducing cellular senescence and decreasing SSPC number and Published under the PNAS license. function. Our data provide convincing evidence that pharmacologic 1To whom correspondence should be addressed. Email: [email protected]. κ inhibition of NF- B activation leads to a functional rejuvenation of This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. the SSPC pool, resulting in bone regeneration equal to that seen in 1073/pnas.1810692116/-/DCSupplemental. young animals.

www.pnas.org/cgi/doi/10.1073/pnas.1810692116 PNAS Latest Articles | 1of10 Downloaded by guest on September 29, 2021 vicinity, commencing a vicious cycle that results in a functional decline of the entire tissue and organ (14, 15). We hypothesized that serum from middle-aged mice contains proinflammatory SASP factors and that this cytokine milieu leads to a functional decline of the skeletal stem cell. SSPCs from young (12-wk-old) mice were exposed to sera from middle-aged (52-wk-old) mice in vitro (Fig. 4A). Compared with the homochronic control group (young serum/young cells), which demonstrated a linear increase in cell proliferation over a 7-d time course, the heterochronic group (middle-aged serum/young cells) exhibited a functional

Fig. 1. Skeletal stem/progenitor cell frequency declines in the aging patient. (A) FACS analysis of ICBG samples from 36 patients (20 male and 16 female) of varying ages revealed a significant (P < 0.05) negative correlation between age and SSPC number. (B) SSPC frequency is significantly decreased in patients older than 50 y of age (P < 0.05). (C) SSPC number is negatively correlated with time to bony union (P < 0.05). Green dots identify fractures that healed clinically and radiographically within 6 mo. Red dots mark patients with fracture union after 6 mo.

C), the injuries in the middle-aged animals exhibited a smaller area of woven bone, with bone formation predominantly between the cortical edges (Fig. 2 B and D). μCT imaging and 3D rendering confirmed this finding (Fig. 2 E and F). Histo- morphometry using μCT demonstrated a smaller callus volume [bone volume/total volume (BV/TV)], trabecular number (Tb. N), and trabecular thickness (Tb.Th) and increased trabecular spacing (Tb.Sp; Fig. 2G). This first experiment demonstrated that 52-wk-old WT mice exhibit a phenotype of age-related impaired bone regeneration. Thus, we elected to use this age group for the subsequent experiments aimed at understanding the effect of aging on bone healing and SSPC function.

Aging Leads to a Decrease in SSPC Number. The key ingredient to successful bone regeneration is the SSPC. To determine whether a decline in SSPC number is responsible for the impaired regenerative capacity of the aging skeleton, as seen in our human cohort, we used FACS with the inclusive SSPC marker LepR − − − + + (12). CD45 CD31 Ter-119 LepR cells (LepR cells) comprise + + + + a heterogeneous mix of Sca-1 , PDGFRα , CD51 , and CD105 SSPCs (SI Appendix, Fig. S1), and Morrison and coworkers (12) + demonstrated that LepR cells make up 0.3% of bone marrow cells; they differentiate into bone, cartilage, and fat in vivo and in vitro and, most importantly, give rise to bone postnatally and in response to injury. Bones from middle-aged mice contained + significantly fewer LepR cells compared with bones from young mice (Fig. 3 A and B), and cfu assays confirmed this finding (Fig. 3C).

Circulating Systemic Factors Lead to Skeletal Stem Cell Aging. Hav- ing now established that SSPC frequency declines in mice similarly to our observation in humans, we next sought to identify the Fig. 2. Aging results in impaired bone healing. (A and B) Histological sec- cause for this decline in stem cell number. Cell senescence, an tions of tibial monocortical defects 14 d after injury in 12-wk-old and 52-wk- irreversible arrest in cell division, has been associated with stem old WT mice stained with Movat’s pentachrome. (C and D) Aniline blue staining depicting bone matrix deposition within the cortical defect. (E and cell attrition in a multitude of other aged tissues (reviewed in ref. μ 13). Cell senescence is accompanied by a senescence-associated F) Three-dimensional CT reconstructions with surface rendering of bony regenerate (red) showing smaller callus size in the defect site of middle-aged secretory phenotype (SASP), a local proinflammatory microen- animals. (G) Analysis of μCT data showing BV/TV (n = 6, P < 0.001), Tb.N (n = vironment, which acts on surrounding cells and inhibits their 6, P < 0.001), Tb.Th (n = 6, P < 0.05), and Tb.Sp (n = 6, P < 0.001) at post- proliferation and cellular function (14). This paracrine effect of operative day (POD) 7 and POD 14 in young and middle-aged mice. bm, the SASP then induces senescence in cells within the immediate bone marrow; c, cortical bone; is, injury site.

2of10 | www.pnas.org/cgi/doi/10.1073/pnas.1810692116 Josephson et al. Downloaded by guest on September 29, 2021 These data strongly suggest that the age-associated decline in SSPC frequency is caused by a systemic proinflammatory environment mediated through NF-κB and likely leading to increased cellular senescence.

NF-κB–Mediated Inflammation Induces SASP in Young Skeletal Stem/ Progenitor Cells. The age-associated cytokine profile leads to a systemic proinflammatory environment, inducing and potentiat- ing NF-κB activation (Fig. 4) (13), and we hypothesize that this inflammatory milieu is responsible for the decrease in SSPC number and function. NF-κB is the fundamental transcriptional regulator of inflammation and controls the expression of genes encoding for proinflammatory cytokines, chemokines, and adhesion molecules (20). Proinflammatory stress and cell senescence activate Nfkb expression (20). We postulate that, with aging, SSPC frequency and function declines, and that this decrease in SSPC number and function is caused by an increased inflammatory microenvironment. To experimentally separate − − inflammation from aging, we used the Nfkb1 / mouse model, which has served as a model organism for low-level chronic inflammation in a plethora of studies involving liver regeneration (21), memory loss (22), and stress response (23). Deletion of NF- κB1 results in the activation of the NF-κB, which in turn leads to − − increased senescence and accelerated aging. Nfkb1 / mice lack the expression of the p105 and p50 NF-κB protein. The lack of κ Fig. 3. SSPC frequency declines with aging. (A) Representative FACS plots of these two NF- B subunits results in the inability to form p50: + young and middle-aged skeletal elements showing decrease in LepR SSPCs p50 homodimers (repressor of proinflammatory gene expression) with aging. (B) Summary plot of FACS data demonstrating decrease in SSPC while still being able to generate RelA-containing NF-κB dimers frequency in 52-wk-old (wo) mice (n = 5, P < 0.01). (C) cfu assay of young and (activators of proinflammatory gene expression), which results in MEDICAL SCIENCES middle-aged bone marrow showing representative colony staining and an enhanced response to inflammatory stimuli (24, 25). Young, − − graphical depiction of quantification (n = 3, P < 0.0001). 30-wk-old Nfkb1 / mice housed in a pathogen-free environment exhibit hallmarks of premature aging with ataxia, kyphosis, sarcopenia, cardiac hypertrophy, and many other age-associated arrest in cell division (Fig. 4B), suggesting the presence of an conditions that are related to an activated chronic inflammatory state “ ” aging factor within the serum. In line with this assumption, (21), thus offering a valuable model organism to study re- β β senescence-associated -gal (SA- -gal) staining revealed a sig- generation in a model of low-grade inflammation in the absence nificant increase in SSPC senescence after heterochronic serum of chronological aging. − − treatment (Fig. 4C). Next, we sought to identify whether the First, we had to confirm that, in fact, 30-wk-old Nfkb1 / mice SSPCs treated with middle-aged serum started to express SASP exhibit a proinflammatory cytokine profile similar to the one factors themselves. Quantitative RT-PCR for SASP markers observed in middle-aged WT mice. qRT-PCR confirmed the demonstrated that, relative to the homochronic control, in- expression of a SASP-like phenotype with up-regulation of Rela terleukin-1α (Il1a), tumor necrosis factor-α (Tnfa), and nuclear (NF-κBp65), Cyclooxygenase 2 (Cox2), Il6, Il10, Tnfa, Il1b, and factor kappa-light-chain-enhancer of activated B cells p65 (Rela) Cdkn2a (p16) in bone marrow stromal and bone-lining cells (Fig. − − significantly increased, consistent with a SASP (Fig. 4D). Thus, 5A). FACS analysis of 30-wk-old, 52-wk-old Nfkb1 / , and age- + aging is associated with a systemic cytokine milieu that directly matched WT mice demonstrated fewer LepR SSPCs in the −/− leads to an arrest of cell division, induces cell senescence, and Nfkb1 mice (Fig. 5B and SI Appendix,Fig.S2A), further con- results in expression of SASP factors. We confirmed these in firming that inflammation, not aging, is driving the decline in SSPC vitro finding by using an SA-β-gal assay (16) on SSPCs freshly number. We next analyzed the expression of SASP factors within harvested from young and middle-aged mice, and we discovered freshly isolated LepR-positive SSPCs and found significantly higher expression levels of Il1b, Il6,andRela in the SSPC population of 30- that middle-aged SSPCs were four times more likely to be se- −/− nescent compared with young SSPCs (Fig. 4 E–G). qRT-PCR of wk-old Nfkb1 mice compared with age-matched WT mice (SI Appendix,Fig.S2B). Gene-expression analysis of the microenvi- the whole bone marrow confirmed an increase in the senescence – markers Cdkn2a (p16) and Cdkn1a (p21) (17–19) (Fig. 4H). ronment, here captured in Q1/2 (CD31, CD45, and Ter-119 posi- In response to the heterochronic serum treatment, we observed tive cells), revealed no changes in comparison with a similar cell population in age-matched WT mice (SI Appendix,Fig.S2B). We an increase in Il1a and Tnfa expression in the young SSPCs (Fig. − − then further investigated the microenvironment of Nfkb1 / mice 4D). These two proinflammatory cytokines lead to activation of and separated the myeloid and lymphoid compartments by using NF-κB, a key mediator of inflammation (20). Therefore, we sur- κ well-accepted surface markers. Within the lymphoid compartment, veyed young and middle-aged SSPCs for NF- B activation. Be- Il1b and Rela were down-regulated and Tnfa was up-regulated in κ − − cause phosphorylated NF- Bp65 (p-p65) is a prerequisite for Nfkb1 / mice, whereas there were no significant differences for κ nuclear localization and thus NF- B activation, we first examined these cytokines in the myeloid compartment (SI Appendix,Fig.S2). κ NF- B activation by using Western blot for p-p65. We observed an These data suggest a shared contribution of the bone marrow and increased p-p65/p65 ratio in middle-aged SSPCs (Fig. 4I). We the SSPC compartment to the proinflammatory milieu to which the then confirmed increased nuclear localization of NF-κBp65 by SSPCs then respond with increased SASP expression (SI Appendix, using immunofluorescence and detected a twofold increase in Fig. S2). Next, we sought to test whether this proinflammatory − − middle-aged SSPCs (Fig. 4 J and K). Together, these data confirm environment in the Nfkb1 / mice inhibits cell division, similar to activation of NF-κB as a key inflammatory mediator in middle- what we had observed in the middle-aged WT mice. We performed aged SSPCs. a cell proliferation assay and detected an absence of cell

Josephson et al. PNAS Latest Articles | 3of10 Downloaded by guest on September 29, 2021 Fig. 4. Systemic cytokines are responsible for aging phenotype. (A) Schematic illustration depicting homochronic (young serum/young cells) and heterochronic (serum from middle-aged mice/young cells) in vitro culture conditions. (B) Cell proliferation assay of young SSPCs treated with sera from young or middle-aged mice showing an inhibitory effect of middle-aged sera on mitotic activity (n = 5). (C) Young SSPCs subjected to middle-aged serum exhibit more cell senescence at 4 d (n = 3, P < 0.01) and 7 d (n = 3, P < 0.001) as measured by SA-β-gal staining. (D) qRT-PCR shows induction of the expression of senescence- associated genes Il1a, Tnfa, and Rela in cells subjected to sera from middle-aged mice (n = 3, P < 0.05). (E–G)SA-β-gal staining of SSPCs from 12- and 52-wk-old mice showing increased senescence (arrowheads) in the aging animal. Quantification reveals a significant increase in senescence at 52 wk of age (n = 4, P < 0.01). (H) Consistent with the SA-β-gal staining, expression levels of Cdkn2a (p16) and cdkn1a (p21) were elevated in the middle-aged bone compartment (n = 7, P < 0.05). (I) Western blot for p-p65 reveals increased NF-κB activation in the middle-aged SSPCs (n = 3, P < 0.05). (J) Immunofluorescence of NF-κBp65 in young and middle-aged SSPCs revealed nuclear localization of NF-κBp65 in the middle-aged cells. (K) Quantification of NF-κBp65 (p65) nuclear localization demonstrates increased NF-κB activation in 52-wk-old SSPCs (n = 3, P < 0.001).

− − proliferation in SSPCs from 30-wk-old Nfkb1 / mice, whereas revealed diminished mineral deposition and alkaline phosphatase − − age-matched WT SSPCs exhibited a linear increase in cell pro- activity in Nfkb1 / cells (Fig. 5E). This decrease in osteogenesis of − − liferation (Fig. 5C). An absence of cell proliferation can be at- the Nfkb1 / cells was confirmed by qRT-PCR for Runx2,osterix tributed to apoptosis, senescence, quiescence, and premature (Osx), and alkaline phosphatase (Alk Phos;Fig.5F). Second, we − − differentiation. We used CellTrace to identify cell-cycle activity analyzed chondrogenic and adipogenic differentiation in Nfkb1 / − − − − of WT and Nfkb1 / cells and demonstrated that Nfkb1 / cells cells. Chondrogenesis, assessed by using micromass cultures, are low-cycling as they are preferentially found in generation 1 revealed smaller and disorganized micromasses without the char- (SI Appendix, Fig. S3 A and B). We then further analyzed the acteristic hypertrophic center surrounded by less differentiated cells within generation 1 and showed that fewer than 1% of cells chondrocytes in the periphery, as seen in the WT micromasses (SI were apoptotic, without difference between the two groups (SI Appendix,Fig.S4A). Adipogenic differentiation revealed an in- Appendix, Fig. S3C). Last, premature differentiation was ruled crease in Oil Red O-positive cells. In addition, we observed a out by using cell-surface markers characteristic for stem cells. greater number of adipocytes in the tibial bone marrow of + + − − We observed no difference in PDGFRα Sca-1 cell number Nfkb1 / mice (SI Appendix,Fig.S4B). Finally, to identify whether − − between the two cell populations (SI Appendix,Fig.S3C). the Nfkb1 / phenotype is truly related to the proinflammatory Thus far, we have shown that the proinflammatory environment environment, we treated young WT SSPCs with serum from young − − − − in Nfkb1 / mice leads to a decrease in SSPC number, an increase Nfkb1 / mice. Serum treatment resulted in a uniform increase in in cell senescence, and a decrease in cell division. Next, we in- proinflammatory cytokine expression in the young cells (SI Ap- − − vestigated the trilineage potential of Nfkb1 / SSPCs. First, we pendix,Fig.S5). The previously described heterochronic serum exposed 30-wk-old SSPCs to osteogenic and growth media (GM) experiments in WT mice (Fig. 4) demonstrated an arrest in pro- and assessed mineral deposition. Whereas WT cells formed con- liferation in response to treatment with serum from middle-aged − − fluent Alizarin red-positive mineral, Nfkb1 / cells deposited only serum (Fig. 4B). We sought to determine whether this same effect small islands of Alizarin red-positive bone matrix when subjected on proliferation can be observed when cells were treated with − − to osteogenic differentiation media (Fig. 5D). Quantification serum from Nfkb1 / mice. We used proliferating cell nuclear

4of10 | www.pnas.org/cgi/doi/10.1073/pnas.1810692116 Josephson et al. Downloaded by guest on September 29, 2021 Fig. 5. Young Nfkb1−/− mice mimic aging phenotype of SSPCs. (A) qRT-PCR of bone tissue from 30-wk-old Nfkb1−/− and age-matched WT mice revealing MEDICAL SCIENCES proinflammatory SASP-like phenotype in Nfkb1−/− mice (n = 3, P < 0.05). (B) Nfkb1−/− mice show characteristic decline of SSPC number at younger age (n = 4, − − − − P < 0.05). (C) Proliferation assay of WT and Nfkb1 / SSPCs showing linear increase in cell number for WT cells and a steady state for Nfkb1 / cells (n = 4). (D) − − Alizarin red staining of mineralization assay for WT and Nfkb1 / cells in osteogenic media (OM) and GM. (E) Quantification of Alizarin red and alkaline − − phosphatase staining showing reduced osteogenic differentiation of Nfkb1 / cells (n = 4, ***P < 0.001). (F) Expression analysis for osteogenic genes of WT − − and Nfkb1 / cells treated with osteogenic differentiation media (n = 4, **P < 0.01 and ***P < 0.001).

antigen (Pcna) gene expression to evaluate cell division in this chronic inflammation, as the expression level returned to levels experiment and observed a decrease in Pcna expression in the cells measured in young animals. − − treated with Nfkb1 / serum (SI Appendix,Fig.S5). Collectively, As we postulated that the age-associated elevation of inflammatory these data confirmed that heightened inflammation, and not cytokines results in increased NF-κB activation, we wanted to de- chronological aging, is responsible for a decrease in number and termine whether the observed systemic NSAID-induced reduction in function of SSPCs. cytokine levels resulted in decreased NF-κB signaling. We again treated young SSPCs with serum from young, middle-aged, and Inflammation-Associated SSPC Decline Is Reversible. Increased cell middle-aged NSAID-treated mice in vitro. This experiment revealed senescence (26), predominant adipogenic differentiation of SSPCs that serum from middle-aged mice treated with sodium salicylate did (27), and amplified apoptosis (28) are all associated with aging and not result in nuclear localization of NF-κBp65, as shown by immu- result in an increased inflammatory response known as inflamm- nofluorescence and quantification (Fig. 6C). We confirmed this ob- aging(29,30).First,wesoughttoidentify whether the inflamm- servation by using Western blot for p-p65, which was increased in the aging phenotype is detectable in middle-aged mice on a systemic cells treated with middle-aged serum and returned to juvenile levels level. We performed multiplex analysis on serum from young and when treated with serum from NSAID-treated middle-aged mice middle-aged mice and detected increases of the proinflammatory (Fig. 6D). cytokines IFN-γ,TNF-α, and IL-6 in the aging animals (Fig. 6A). Previously, we had shown that the proinflammatory environ- We confirmed this finding by qRT-PCR, which revealed up- ment had a direct negative effect on cell division (Fig. 4B)and induced senescence (Fig. 4 C and E–G). If salicylate treatment regulated expression of the proinflammatory cytokines Rela, Tnfa, reduces this chronic inflammatory milieu, theoretically, cell se- Il6, Il1a,andIl1b in SSPCs from middle-aged animals (Fig. 6B). nescence should decrease as a result. We performed an SA-β-gal These experiments confirmed that 52-wk-old WT mice exhibit an assay with cells from young, middle-aged, and middle-aged inflammatory cytokine profile consistent with inflamm-aging. If, in salicylate-treated mice. Similar to our previous results (Fig. 4G), fact, chronic low-grade inflammation is responsible for the decrease with aging, the senescent cell fraction increased (Fig. 6E); in SSPC number, increased senescence, and overall decreased however, after salicylate treatment, the percentage of SA-β-gal– regenerative potential of aged animals, treatment with an antiin- positive cells significantly decreased (Fig. 6E). This was con- flammatory drug may overcome these negative effects. We there- firmed in the heterochronic serum assay. Young SSPCs treated fore treated 52-wk-old mice with sodium salicylate, a low-grade with serum from middle-aged antiinflammatory-treated mice exhibi- antiinflammatory agent proven to inhibit NF-κB pathway activation ted a senescence phenotype similar to the young homochronic group (31–33), for 8 wk, and then harvested SSPCs and analyzed the ex- (SI Appendix,Fig.S6). If senescence is reduced in response to salic- pression levels of the aforementioned pro- and antiinflammatory ylate treatment, does this lead to an increase in the SSPC frequency cytokines. Salicylate treatment resulted in a decrease of the proin- within the bone marrow? By using FACS for LepR, we showed that flammatory cytokines Rela, Cox2,andIl1b (Fig. 6B). This confirmed aging resulted in a significant decrease in SSPC number and that that the treatment protocol successfully repressed aging-induced salicylate treatment partially recovered this loss of SSPC number (Fig.

Josephson et al. PNAS Latest Articles | 5of10 Downloaded by guest on September 29, 2021 Fig. 6. Antiinflammatory treatment reverts inflamm-aging phenotype and increases SSPC pool. (A) Serum cytokine levels of IFN-γ (P < 0.05), TNF-α (P < 0.01), and IL-6 (P < 0.01) in young and middle-aged WT mice (n = 10). (B) Antiinflammatory drug treatment with salicylate reverses inflamm-aging phenotype (n = 6, P < 0.05). (C) Immunofluorescence for NF-κBp65 reveals nuclear localization of NF-κBp65 after treatment with middle-aged serum and a decrease in nuclear localization of NF-κBp65 in cells subjected to serum from middle-aged NSAID-treated mice. Quantification confirms increase in NF-κB activation with aging and decrease to juvenile levels after NSAID treatment (n = 4, P < 0.01). (D) Western blot for p-p65 confirming the decreased NF-κBactivationinresponsetoNSAID treatment (n = 3, *P < 0.05 and **P < 0.01). (E)SA-β-gal staining of young, middle-aged, and salicylate-treated middle-aged SSPCs show reversal of senescence phenotype in aging animals (n = 3, P < 0.05). (F) Graph showing SSPC frequency in young, middle-aged, and salicylate-treated middle-aged mice (n = 11, **P < 0.01 and ***P < 0.001). (G) Cfu-forming assay confirms increase of SSPC frequency after salicylate treatment (n = 3, P < 0.001). tx, treated; wo, weeks old.

6F). Cfu-forming assay analysis confirmed the reversal of decreased stark separation between young and middle-aged SSPCs (Fig. 7A). cfu formation after salicylate treatment (Fig. 6G). These analyses In line with our FACS analysis showing a rescue of SSPC frequency, further confirmed that it is the inflammatory component of we observed a shift of the transcriptional profile of middle-aged inflamm-aging and not the chronological aging component that SSPCs to that of the young SSPCs after antiinflammatory treat- leads to a decrease in SSPC number. ment. We then used gene-set enrichment analysis (GSEA) to further understand this potential rejuvenation of the SSPC pool (Fig. RNA Sequencing Analysis Reveals Rejuvenation of the SSPC Pool After 7B). SSPCs from young animals enriched for genes associated with Antiinflammatory Treatment. Number and function of SSPCs are critical for successful bone regeneration. Having established that stemness, as did SSPCs from middle-aged antiinflammatory-treated SSPC number declines with age and that this decrease can be halted mice, again supporting a reversal of the aging phenotype. Similarly, by modulating the age-associated proinflammatory environment, we genes associated with osteogenesis and decreased adipogenesis next sought to understand the effect of changes in the immune were enriched in the young SSPCs, and again immunomodulation environment on the transcriptome of SSPCs. We first used an un- with an antiinflammatory agent led to an increased enrichment for biased sequencing approach to compare the transcriptome of young these gene sets in middle-aged SSPCs (Fig. 7B). From these data, and middle-aged SSPCs. Hierarchical cluster analysis revealed a we conclude that, on a transcriptional level, modulation of the

6of10 | www.pnas.org/cgi/doi/10.1073/pnas.1810692116 Josephson et al. Downloaded by guest on September 29, 2021 function of the rejuvenated SSPC in an in vitro and in vivo environment. First, we evaluated the bone marrow compartment as a whole. Gene expression levels of Osx and Osteocalcin (Oc) significantly decreased in response to aging (Fig. 8A). We next analyzed bone marrow cells from middle-aged mice treated with salicylate, and here Osx, Oc,andAlp significantly increased compared with middle-aged untreated animals, and even reached levels equal to or higher than cells from young animals (Fig. 8A). To test the functional differentiation capacity of SSPCs, we exposed SSPCs to osteogenic differentiation media in vitro and then quantified mineralization as a readout for osteogenic differentiation. Young SSPCs demonstrated robust mineralization, whereas middle-aged SSPCs showed only some isolated foci of mineralization (Fig. 8B). SSPCs harvested from salicylate-treated middle- aged mice recovered their osteogenic function, which resulted in mineralization comparable to that of young mice (Fig. 8B). Aging has been associated with fatty degeneration of the bone marrow compartment (27). To directly test whether inflammation associated with aging can be attributed to this fatty degeneration, we subjected young and middle-aged SSPCs to adipogenic differentiation media. As expected, middle-aged cells were more prone to differentiate into adipocytes, as shown by increased Pparg and Fabp4 expression, and this was reversed in cells from NSAID- treated mice (Fig. 8C). We confirmed this expression pattern by using a functional adipogenic differentiation assay and again demonstrated an increase in adipogenesis with aging (Fig. 8D). However, SSPCs from middle-aged salicylate-treated mice exhibited less adipogenic differentiation (Fig. 8D), suggesting that inflamm- aging plays an active role in fatty degeneration of the bone marrow. MEDICAL SCIENCES We then evaluated tissue sections of young, middle-aged, and middle-aged NSAID-treated mice and observed a significant increase in adipocyte number with aging, which was reversed in mice treated with sodium salicylate (Fig. 8E). Next, we investigated whether the suppression of low-grade inflammation had a direct effect on homeostasis of the skeleton and bone regeneration in vivo. First, we analyzed bone homeostasis and performed μCT analysis of lumbar spine segments and humeri of young, middle-aged, and middle-aged salicylate- treated mice. BV/TV, Tb.N, and Tb.Th were significantly reduced in aged animals compared with young animals, whereas Tb.Sp was increased, confirming an age-appropriate bone loss (SI Appendix, Fig. S7). Middle-aged animals treated with salicylate showed a skeletal phenotype comparable to the untreated middle-aged control animals, indicating that salicylate treatment over a 12-wk time course in this age group did not affect bone homeostasis. Finally, we set out to examine whether the decrease in cell senescence, increase in SSPC number, and shift of the osteogenic/ adipogenic balance toward osteogenesis in response to antiin- Fig. 7. RNA-seq analysis reveals a shift toward increased stemness and osteo- flammatory treatment resulted in a measurable proregenerative genesis and decreased adipogenesis in middle-aged antiinflammatory-treated effect in vivo. To avoid masking effects of an endogenous healing mice. (A) Heat map representing gene-expression values of the top 133 genes response in a skeletal injury, we elected to use an ectopic trans- with a false discovery rate-adjusted P value less than 0.01 (q > 0.01)acrossall plantation model, as this represents the most stringent readout of in samples. Hierarchical clustering of these genes reveals that LepR+ SSPCs isolated vivo bone formation of SSPCs (34). Here, we used a subrenal + from 52-wk-old antiinflammatory-treated mice cluster with 12-wk-old LepR capsule transplantation assay to test whether suppression of chronic SSPCs. Columns indicate single samples, and rows indicate genes. (B)GSEAplots inflammation in the middle-aged animal successfully restores oste- demonstrate that young SSPCs and middle-aged treated SSPCs positively cor- ogenic capacity of the SSPCs in an in vivo setting. The subrenal relate with gene sets for stemness (BOQUEST STEM CELL UP), osteogenesis (SKELETAL DEVELOPMENT), and decreased adipogenic potential (TSENG ADI- capsule assay represents an ideal functional assay because cells POGENIC POTENTIAL DN). transplanted between the renal capsule and the parenchyma receive sufficient blood supply and nutrients while being devoid of proos- teogenic or prochondrogenic stimuli that would confound the proinflammatory environment in the aging animal leads to a re- readout (35). SSPCs from young, middle-aged, and middle-aged versal of the aging phenotype. salicylate-treated mice were transplanted under the renal capsule. After 3 wk, histological staining and histomorphometry revealed a Antiinflammatory Drug Treatment Abrogates the Aging Phenotype of decrease in bone formation in the group containing middle-aged SSPCs and Restores Regenerative Potential. The previous set of ex- SSPCs compared with young SSPCs (Fig. 8 F and H). In stark periments using unbiased sequencing strongly suggest that SSPC contrast, cells transplanted from salicylate-treated middle-aged mice aging can be reversed. Next, we set out to interrogate the cellular exhibited a robust osteogenic response, similar to that observed with

Josephson et al. PNAS Latest Articles | 7of10 Downloaded by guest on September 29, 2021 Fig. 8. SSPC function is restored in aging animals after antiinflammatory treatment. (A) qRT-PCR of SSPCs shows increased osteogenic gene expression after repression of inflamm-aging (n = 3, *P < 0.05, **P < 0.01, and ***P < 0.001). (B) Representative images and quantification of osteogenic differentiation analyzed by Alizarin red staining (n = 3, P < 0.001). (C) Adipogenic differentiation analyzed by qRT-PCR for adipogenic markers (n = 3, P < 0.05) and (D)Oil Red O staining (representative images and quantification; n = 3, **P < 0.01 and ***P < 0.001). (E) Histomorphometry for adipocyte number in the tibial bone marrow of young, middle-aged, and middle-aged NSAID-treated mice (n = 5, *P < 0.05 and ***P < 0.001). (F and G) Pentachrome histology of renal capsule (rc) transplants of 52-wk-old (wo) untreated and salicylate-treated SSPCs into young WT host mice. (F) Histomorphometry shows a decrease in regenerate size in middle-aged mice and restoration of regenerative function after salicylate treatment (n = 4, P < 0.05). bg, bone graft; tx, treated.

juvenile SSPCs (Fig. 8 G and H), indicating that suppression of the described approach resulted in an inflammatory milieu com- chronic inflammation in the middle-aged animal can restore the parable to that of a young animal, thus allowing us to study whether regenerative capacity of SSPCs. the detrimental effects of inflamm-aging on SSPCs can be reversed. The effect of inflamm-aging on SSPCs can be broken up into Discussion cell-intrinsic and cell-extrinsic changes. Whereas the proinflammatory Chronic inflammation in elderly subjects has been linked to a environment of the aged skeleton exerts its negative effect on the variety of diseases (20). Here, we provide evidence that SSPCs stem/progenitor cell through cell-extrinsic mechanisms such as respond to elevated levels of proinflammatory cytokines with modulation of signaling pathways [Wnt, Notch, BMPs (37)], cell- increased senescence, decreased stem/progenitor cell number, intrinsic mechanisms include self-renewal defects and induction of and decreased functionality. In a pharmacological rescue experi- stress-induced pathways that lead to cellular senescence (13). ment, we show that reduction of age-related chronic inflammation Whereas cell-intrinsic defects such as senescence and self-renewal leads to a functional restoration of bone regeneration through a defect are considered irreversible, extrinsically induced defects may decrease in stem/progenitor cell senescence, increase in stem/pro- be reparable. As shown here, modulation of the inflammatory milieu genitor cell number, and osteogenic gene expression (Fig. 9). by using a pharmacological compound resulted in a restoration of Chronic inflammation in elderly subjects has been attributed regenerative capacity. Interestingly, we observed that a short expo- to a constant decay of extracellular macromolecules and in- sure of middle-aged SSPCs to a young systemic environment, as seen tracellular organelles, resulting in an initiation and maintenance in the renal capsule transplantation assay, did not lead to a functional of an immune response (29). Degeneration of aged cells leads to restoration. This suggests that the brief exposure to the young envi- secretion of reactive molecules, proinflammatory enzymes, and ronment alone is not sufficient to functionally rejuvenate SSPCs or mediators that in turn will lead to further deterioration of the that the molecular microenvironment repressed the properties of the tissue, starting a vicious cycle that is characteristic of this chronic resident stem/progenitor cells, and, when modulated with an antiin- condition. Although all cells are affected by this proinflammatory flammatory drug, this repression was reversed, resulting in return of milieu, the impact on the resident stem/progenitor cell, responsible regenerative potential. This finding is supported by heterochronic for regeneration in response to injury, is likely the most detrimental, transplantation assays and parabiosis experiments, which showed that as it jeopardizes maintenance of tissue integrity. The toxic media- the aging of a variety of stem cells was largely driven by cell-extrinsic tors within the tissue environment result in DNA damage, protein mechanisms of the surrounding environment (38). In these assays, degradation, and organelle injury of the stem/progenitor cell, introduction of circulation from young animals restored the re- causing cellular senescence, which in turn results in further stimu- generative potential of the aged skeleton (39), indicating that the lation of the chronic inflammatory status through the cell’ssecretory detrimental effects of aging on the progenitor pool may be reversible phenotype known as the SASP (29, 36). Here, we aimed at dis- (40). A prime target for the mechanism of action observed in the rupting this vicious cycle by modulating the proinflammatory envi- parabiosis model is the inflammatory cytokine environment. Here, we ronment by using a mild antiinflammatory drug. As shown by serum provide strong evidence that inflammation, not chronological aging, cytokine analysis and gene-expression analysis of bone marrow cells, is the main driver of SSPC dysfunction, and we demonstrate that

8of10 | www.pnas.org/cgi/doi/10.1073/pnas.1810692116 Josephson et al. Downloaded by guest on September 29, 2021 orthopedic conditions, the baby-boomer generation (45). It is this population that would most benefit from a translational approach that improves bone regeneration. Septa- and octogenarians, on the contrary, often undergo replacement procedures (i.e., hip hemi- arthroplasty, shoulder arthroplasty) rather than being subjected to procedures that rely on functional bone regeneration. Thus, our study was executed by using 52-wk-old mice in an attempt to simulate the physiology of middle-aged orthopedic patients. Sodium salicylate, an antiinflammatory drug with similar antiinflammatory potency as aspirin, exerts its effect through indirect inhibition of prostaglandin biosynthesis (reviewed in ref. 46). Although this nonspecific inhibitory effect undoubtedly reversed the inflamm-aging phenotype of the SSPCs in these murine models, future work will focus on a more targeted suppression of inflamm- aging using specific Cox2 inhibitors, small-molecule antagonists against epigenetic regulators of the innate immune system, and modulators of NF-κB activity. In summary, we present compelling data that age-associated inflammation, regulated by NF-κB and triggered by increased cell senescence, leads to a functional decline of SSPCs, which can Fig. 9. Schematic illustration of the cellular mechanism leading to impaired be overcome and reversed by suppression of inflammation by bone regeneration in elderly subjects. During aging, senescent SSPCs secrete using a low-grade antiinflammatory drug. SASP factors, which result in activation of NF-κB in adjacent SSPCs, inducing them to undergo senescence. This accumulation of senescent SSPCs leads to Materials and Methods decreased SSPC self-renewal and proliferation, resulting in a decrease in Patients and Specimens. All experiments involving human subjects were ap- overall number. Together with a decline in osteogenic differentiation, this proved by the New York University (NYU) School of Medicine Institutional leads to impaired bone regeneration in elderly subjects. NSAID treatment Review Board. After informed consent was obtained, specimens were κ inhibits NF- B activation, thereby blocking the effect of the SASP factors on obtained during routine ICBG harvest. One cubic centimeter of ICBG was surrounding SSPCs, which leads to a decrease in SSPC senescence, increase in immediately transferred into a microcentrifuge tube and placed on ice. MEDICAL SCIENCES SSPC number, improved osteogenic differentiation, and, finally, enhanced Samples were dissociated and stained with antibodies against CD45 and bone regeneration. CD271. FACS analysis was performed by using a BD LSRII cell analyzer with high-throughput sampler. CD45-negative and CD271-positive SSPCs were displayed as percentages of total single cell number (8–11). Patient data, NF-κB activation in young animals can mimic the aging phenotype in including radiographic and clinical time to union, were extracted from a respect to SSPC number and function. This is further supported by prospective patient database at the NYU Langone Orthopedic Hospital. the expression of the senescence-associated genes Cdkn1a (p21) and Cdkn2a (p16). Recent data suggest distinct roles for Cdkn1a and Isolation of SSPCs. Tibiae and femurs were harvested as previously described Cdkn2a. Kim et al. (41) and Baker et al. (42) suggest that expression (47). Dissociated cell samples were stained with antibodies against CD31, CD45, Ter-119, and LepR for purification by flow cytometry (Moflo XDP; of Cdkn1a is associated with senescence induced by chronological − − − + aging, whereas Cdkn2a expression is associated with stress-induced Beckman-Coulter). CD31 CD45 Ter-119 LepR cells were identified as SSPCs (12, 34). senescence (43). In our experiments, dysfunctional SSPCs from middle-aged mice exhibit heightened Cdkn2a expression (Figs. 4H Isolation and Culture of SSPCs. For the in vitro experiments, tibial and femoral and 5A), possibly indicating a stress response to the increased in- SSPCs were isolated by centrifugation (48). SSPCs were resuspended in GM flammatory environment rather than an age effect. (DMEM containing 10% FBS and 1% penicillin/streptomycin; Thermo Fisher Our findings provide strong support for a central role of NF-κB Scientific) and then plated in 75-mL tissue culture flasks. Media was changed as a mediator of inflamm-aging. Chen et al. (44) previously every 2 d. All cellular assays described were performed with SSPCs at passage demonstrated a link between aging and NF-κB activation in a 1 from at least three different mice in three technical replicates. progeroid mouse model during bone homeostasis, supporting our findings during bone regeneration. During homeostasis, NF-κB– Statistical Analysis. A priori power analysis to obtain statistical significance = = mediated inflammation promotes osteoclastogenesis (44) in the (P 0.05, power 80%) resulted in n 4 for each group after body-size adjust- aging animal, which may contribute to the osteoporotic phenotype ment, expecting a 25% difference between the groups. Prism 7 (GraphPad Software) was used for statistical computations. A Student’s t test was used for characteristic of aging animals. Although osteoclasts play an im- all comparisons in which there were two groups; ANOVA followed by Holm– portant role during regeneration, their main function is centered Sidák correction for post hoc testing was applied for analyses in which there around the remodeling phase of repair, which occurs at later were two or more comparisons being made. Error bars in the figures represent stages when bone matrix deposition has been completed. Because SEMs. An asterisk denotes a P value <0.05 unless denoted otherwise in the our focus aimed at the early stages of regeneration rather than figure legend. remodeling and homeostasis, we did not further examine the Further standard materials and methods including animals, connection between NF-κB and osteoclastogenesis. antiinflammatory treatment, monocortical defects, renal capsule transplants, his- μ There are caveats to this work. Usually, 52-wk-old mice are tology and histomorphometry, CT analyses, multiplex ELISA, SDS/PAGE and not considered aged yet; however, as demonstrated here, they Western blots, cfu assay, proliferation assay, osteogenic and adipogenic differentiation, RNA isolation and quantitative real-time PCR, in vitro clearly exhibit an increased proinflammatory cytokine profile homochronic and heterochronic serum treatment, identification of senes- consistent with the process of aging. We selected this age group cent cells, cell-cyle analysis, and RNA sequencing (RNA-seq) analysis are rather than studying older animals for two reasons. First, SSPC described in the SI Appendix. number is already significantly decreased at 1 y of age, rendering transcriptional analyses of this cell population difficult. At 2 y, ACKNOWLEDGMENTS. We thank Ripa Chowdhury (NYU College of Den- when mice are considered aged, SSPC number is very low, tistry) for assistance with the μCT imaging, funded through NIH Grant S10 OD010751. Cell sorting/flow cytometry technologies were provided by NYU making it impossible to study the effects of inflamm-aging on this Langone’s Cytometry and Cell Sorting Laboratory, and RNA-seq and analysis cell population. Second, we chose this middle-aged group to was performed in the Genome Technology Center, both of which are sup- better represent the human age cohort most often debilitated by ported by NIH/National Cancer Institute Grant P30CA016087. This work was

Josephson et al. PNAS Latest Articles | 9of10 Downloaded by guest on September 29, 2021 supported by NIH/National Institute on Aging Grant 1R01AG056169; NIH/ Education Foundation and the Orthopaedic Trauma Association, funded National Institute of Arthritis and Musculoskeletal and Skin Grant in part by Zimmer Biomet, Depuy Synthes, and the Society of Military K08AR069099 (to P.L.); and a grant from the Orthopaedic Research and Orthopaedic Surgeons.

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